This article aimed to explore the targets of the innovative drug HSK31679 in treating metabolic dysfunction-associated steatohepatitis (MASH), and to validate the changes in drug efficacy before and after target knockout through in vivo experiments.

Using the drug and disease databases, HSK31679 and MASH related targets were identified. The core targets were further determined by Cytoscape software. Auto Dock predicted the binding affinity of corresponding proteins to HSK31679. MASH mouse models of wild-type and liver caspase 8 (CASP8) knockout were constructed. Mice were divided into control group (normal feeding of mice + 0.5 mL saline administration), model group (modeling of mice + 0.5 mL saline administration), experimental group (modeling of mice + 3 mg·kg^-1 HSK31679 administration), ΔCASP8 model group (modeling of CASP8 knockout mice + 0.5 mL saline administration) and ΔCASP8 experimental group (modeling of CASP8 knockout mice + 3 mg·kg^-1 HSK31679 administration), with 8 mice in each group. Serum biochemical indicators were measured using an enzyme-linked immunosorbent assay (ELISA) after treatment, and liver sections were stained with HE to velidate the role of core targets of HSK31679′s treatment of MASH.

HE staining revealed significant and typical MASH-associated cellular damage in hepatocytes of the model group, indicating successful model establishment. Five major core targets associated with both HSK31679 and MASH were identified, and HSK31679 exhibited favorable binding activity with the CASP8 protein among them. In animal experiments, the serum triglyceride (TG) levels in the blank group, model group, experimental group, ΔCASP8 model group, and ΔCASP8 experimental group were (0.61±0.05), (1.01±0.15), (0.66±0.13), (0.62±0.09), and (0.63±0.12) mmol·L^-1, respectively; the total cholesterol (TC) levels were (1.24±0.13), (2.88±0.32), (1.60±0.29), (1.30±0.35), and (1.32±0.10) mmol·L^-1, respectively; the alanine aminotransferase (ALT) levels were (74.64±9.31), (140.20±23.74), (88.67±13.67), (78.18±17.66), and (73.58±16.15) IU·L^-1, respectively; the aspartate aminotransferase (AST) levels were (106.33±26.77), (227.32±20.51), (124.58±13.63), (138.37±18.08), and (115.54±27.14) IU·L^-1, respectively. Compared with the model group, the differences of above indicators in the experimental group were all statistically significant (all P<0.01). In HE staining, hepatocytes were arranged in a relatively orderly and dense manner, with a significant reduction in cytoplasmic vacuoles. The aforementioned indicators in the ΔCASP8 experimental group showed no statistically significant differences compared to the ΔCASP8 model group (all P>0.05). There was also no significant difference in the degree of hepatocyte degeneration between the two groups.

CASP8 protein is the key target of HSK31679 in the treatment of MASH. HSK31679 alleviated MASH-related hepatic inflammatory symptoms by binding to CASP8 protein and reducing its activity.